CN103409515A - Kit used for species identification of channel catfish and application method of kit - Google Patents
Kit used for species identification of channel catfish and application method of kit Download PDFInfo
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Abstract
The invention relates to a kit used for species identification of channel catfish and an application method of the kit. The kit comprises following reagents which are packaged separately: a lysis liquid, extract liquid I, extract liquid II, a PCR reaction liquid, an enzyme digestion reaction liquid, a negative control reagent and a positive control reagent. The application method comprises steps of DNA extraction, PCR amplification and enzyme digestion identification. The kit and the application method are capable of identifying channel catfish species at a molecular level; operation is simple and easy; identification is fast; the kit and the application method are practical; consumed time is short; the channel catfish species can be identified quickly and conveniently based on both integral channel catfish body and processed finished products or semi-finished products; and the kit and the application method are suitable for rapid species identification of channel catfish in import and export trade.
Description
Technical field
The present invention relates to a kind of test kit and using method thereof, relate in particular to a kind of channel catfish species identification kit and using method thereof, belong to molecular biology PCR Genetic identification field.
Background technology
Channel catfish (Ictalurus punctatus), also claim ditch Nian (Channel Catfish), and (Siluriformes), Channel-catfish section (Ictaluridae), Cha Wei Channel-catfish belong to (Ictalurus) to belong to Silurformes.It originates in North America, and its NATURAL DISTRIBUTION district is at cen.am., Canadian south and northern Mexico.The expensive economic freshwater fish of channel catfish generic name, be subjected to liking of the U.S., Canada and other many national human consumers deeply.The finished product processed or work in-process all are in great demand in the U.S., Japan, Europe and the market, ground such as Canadian very much, and market demand is very big.From 1984, China Hubei Province aquatic products institute introduces channel catfish first, along with breeding, the reaching its maturity of cultivation and processing technology, add the investment repayment benefit of great number, make aquaculture and the processing industry of channel catfish flourish rapidly in China, the foreign exchange earning benefit is also soaring year by year.
In recent years, once Vietnam bar shark that was considered as " Antidumping " by United States Government and was closed down is being labeled the American market that enters of " Chinese channel catfish ", this is the brand of grievous injury China channel catfish not only, affected the normal exit of China's channel catfish product, and having upset international channel catfish market, Enterprises for Export in China is not repeatedly returned goods and is suffered tremendous economic loss because by the U.S., being inconsistent to serve as reasons with species.The inspection and quarantining for import/export department that ensures economic trade development as main support technological means has also faced a brand-new task and challenge.
For manufactured channel catfish finished product and work in-process, because form has occurred to change fully, traditional species authentication method of Main Basis skeletal structure and formalness feature is identified fully inapplicable at the species of channel catfish.In addition, in finished product and work in-process, due to the serious sex change of cyto-architectural destruction and protein, identify according to the cytotaxonomy method of karyological character and according to the species that the zymetology authentication method of isozyme data etc. is not suitable for channel catfish too.
Along with reaching its maturity of round pcr and DNA sequencing technology, the genetic variation and genetic differentiation of understanding between species by DNA sequence dna information becomes more and more general.Mitochondrial DNA is fast with its evolutionary rate, follows the characteristics such as matrilinear inheritance, becomes one of significant notation of population genetics research.Relevant science and technology is looked into new result and is shown, the data of the PCR-RFLP of external existing Mitochondrial DNA are carried out the report that in the goods such as cod, flatfish, species are identified, but all do not have the molecular data in the D-loop district of Mitochondrial DNA to carry out the report of channel catfish species evaluation aspect.And the operating process of PCR-RFLP method is loaded down with trivial details, multiplexly in the different groups to same species, carry out analysis of genetic polymorphisms, be unsuitable for quick, economic evaluation channel catfish species.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of channel catfish species identification kit and using method thereof, solves the deficiencies in the prior art, utilizes manufactured channel catfish finished product and work in-process, identifies fast and easily the channel catfish species.
The technical scheme that the present invention solves the problems of the technologies described above is as follows: a kind of channel catfish species identification kit comprises the reagent that following separation is packed: lysate, extraction liquid I, extraction liquid II, PCR reaction solution, endonuclease reaction liquid, negative control and positive control;
Described lysate pH value is 8, comprises following component: 200mmol/L NaCl, 20mmol/LTris-HCl, 50mmol/L EDTA, 1%SDS, and all the other are water;
Described extraction liquid I comprises phenol, chloroform and primary isoamyl alcohol, and wherein the volume ratio of phenol, chloroform and primary isoamyl alcohol is 25:24:1;
Described extraction liquid II comprises chloroform and primary isoamyl alcohol, and wherein the volume ratio of chloroform and primary isoamyl alcohol is 24:1;
Described PCR reaction solution comprises following component: 10 * PCR Buffer, MgCl
2, dNTPs, upstream primer, downstream primer, Taq archaeal dna polymerase, all the other are ultrapure water; Concrete primer sequence is:
Upstream primer: 5 '-GTCGCCTCCGTACTGTATTTCTCC-3 ',
Downstream primer: 5 '-GGGTCCATCTTAACATCTTCAGTG-3 ';
Described endonuclease reaction liquid comprises 10 * Buffer and EcoR I;
Described positive control be after pcr amplification enzyme cut the channel catfish D-loop standard model identified (being that known channel catfish species D-loop carries out the product result that pcr amplification and enzyme obtain after cutting);
Described negative control is the channel catfish D-loop standard model cut of enzyme (be known channel catfish species D-loop carry out pcr amplification do not carry out enzyme and cut the product result obtained) not after pcr amplification.
On the basis of technique scheme, the present invention can also do following improvement.
Further, in described PCR reaction solution, contain 10 * PCR Buffer2.5 μ l, MgCl
22.0 μ l, dNTPs2.0 μ l, upstream primer 0.5 μ l, downstream primer 0.5 μ l, Taq archaeal dna polymerase 0.5 μ l, and ultrapure water 16 μ l.
Further, in described endonuclease reaction liquid, contain 10 * Buffer2.0 μ l and EcoR I 1.0 μ l.
Further, described lysate room temperature is deposited, and described extraction liquid I and extraction liquid II are stored in 4 ℃, and PCR reaction solution, endonuclease reaction liquid, negative control and positive control are stored in-20 ℃.
Another technical scheme that the present invention solves the problems of the technologies described above is as follows: a kind of using method of channel catfish species identification kit comprises the following steps:
Step 1, DNA extraction:
1) get sample 0.08～0.12g to be detected, after fully pulverizing, add 500 μ l lysates, be placed in 50～60 ℃ of water-baths and hatch 1h～2h, obtain mixed solution;
2) in mixed solution, add equal-volume extract I, carry out the centrifugal 10min of 12000r/min, get supernatant liquor;
3) in supernatant liquor, add equal-volume extract I, carry out the centrifugal 10min of 12000r/min, get supernatant liquor;
4) in supernatant liquor, add equal-volume extract II, carry out the centrifugal 10min of 12000r/min, get supernatant liquor;
5) in supernatant liquor, add the NaAC of 1/10 volume and the dehydrated alcohol of 2 times of volumes, mix gently, in-20 ℃ of precipitation 1h, then carry out the centrifugal 10min of 12000r/min;
6) solution after centrifugal is cleaned once with the ethanol that volume fraction is 70%, the more centrifugal 5min of 10000r/min, remove supernatant liquor, be precipitated;
7) will precipitate natural air drying, and add 100 μ l sterilized waters and dissolve, obtain DNA solution, standby;
1) the PCR reaction solution that thaws on ice, make it to dissolve fully, and the PCR reaction solution is joined in the PCR reaction tubes;
2) get the DNA solution 1 μ l that step 1 obtains, add in the PCR reaction tubes, mix;
3) the PCR reaction tubes is put into to the PCR instrument, response procedures is as follows: 95 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, annealing 30s, 72 ℃ are extended 1min, 35 circulations, last 72 ℃ are extended 10min, then are cooled to 12 ℃;
4) the PCR product of gained is placed on ice and can uses, or be placed in-20 ℃ standby;
1) get step 2 and obtain PCR product 5 μ l, mix with endonuclease reaction liquid, 37 ℃ of incubation 2～3h, obtain enzyme and cut product;
2) above-mentioned enzyme is cut to product and positive control and negative control and carried out agarose gel electrophoresis, take and identify whether testing sample is channel catfish according to electrophoresis result.
The invention has the beneficial effects as follows: mitochondrion DNA control area has individuality and group specificity, therefore be used as widely in the world the standard of species evaluation, especially D-loop control region, different plant species differs greatly, can the precise Identification species, on the basis to the D-loop order-checking, find its restriction enzyme site, and carry out enzyme and cut evaluation, do not need order-checking can identify simply the channel catfish species.Test kit of the present invention can be quick and easy complete above-mentioned steps, from molecular level, identify the channel catfish species, operation is simple, Fast Practical, spent time is of short duration, extremely is applicable to channel catfish species in foreign trade are carried out to Rapid identification.
The accompanying drawing explanation
Fig. 1 is chance sample of the present invention and positive control, negative control agarose gel electrophoresis figure, the wherein negative contrast of c1, the negative contrast of c2,10,27,30,34,42 sample number into spectrum for choosing at random.
Embodiment
Below in conjunction with accompanying drawing, principle of the present invention and feature are described, example, only be used to explaining the present invention, is not intended to limit scope of the present invention.
A kind of channel catfish species identification kit, each test kit can detect 10 samples, and described test kit comprises the reagent of following separation packing: lysate, extraction liquid I, extraction liquid II, PCR reaction solution, endonuclease reaction liquid, negative control and positive control;
Described lysate pH value is 8, comprises following component: 200mmol/L NaCl, 20mmol/LTris-HCl, 50mmol/L EDTA, 1%SDS, and all the other are water; The each 500 μ l that use of described lysate, contain altogether 5ml in each test kit; Room temperature is deposited.
Described extraction liquid I comprises phenol, chloroform and primary isoamyl alcohol, and wherein the volume ratio of phenol, chloroform and primary isoamyl alcohol is 25:24:1; 50ml phenol, 48ml chloroform and 2ml primary isoamyl alcohol are mixed, obtain 100m and extract the liquid I; Described extraction liquid I is used 1ml at every turn, in each test kit, contains altogether 10ml; 4 ℃ of storages;
Described extraction liquid II comprises chloroform and primary isoamyl alcohol, and wherein the volume ratio of chloroform and primary isoamyl alcohol is 24:1; 96ml chloroform and 4ml primary isoamyl alcohol are mixed, obtain 100m and extract the liquid II; The each 500 μ l that use of described extraction liquid II, contain altogether 5ml in each test kit; 4 ℃ of storages;
Described PCR reaction solution comprises following component: 2.5 μ l10 * PCR Buffer, 2.0 μ l MgCl
2, 2.0 μ l dNTPs, 0.5 μ l upstream primer, 0.5 μ l downstream primer, 0.5 μ l Taq archaeal dna polymerase, and ultrapure water 16 μ l; The each 24 μ l that use of described PCR reaction solution, contain altogether 240 μ l in each test kit;-20 ℃ of storages;
Concrete primer sequence is:
Upstream primer: 5 '-GTCGCCTCCGTACTGTATTTCTCC-3 ',
Downstream primer: 5 '-GGGTCCATCTTAACATCTTCAGTG-3 ';
Described endonuclease reaction liquid comprises 2.0 μ l10 * Buffer and 1.0 μ l EcoR I, and the each 3 μ l that use of described endonuclease reaction liquid, contain altogether 30 μ l in each test kit;-20 ℃ of storages;
Described positive control be after pcr amplification enzyme cut the channel catfish D-loop standard model of identifying, the each 5 μ l that use of described positive control, contain altogether 50 μ l in each test kit;-20 ℃ of storages;
Described negative control is the channel catfish D-loop standard model cut of enzyme not after pcr amplification, and the each 5 μ l that use of described negative control, contain altogether 50 μ l in each test kit;-20 ℃ of storages.
Choose at random 50 of channel catfish work in-process to be exported, sample is numbered to 1～50, choose at random 5 samples, carry out following experiment.
Embodiment 1
Step 1, DNA extraction: get sample 0.1g to be detected No. 10, after fully pulverizing, add 500 μ l lysates, be placed in 53 ℃ of water-baths and hatch 1.5h, obtain mixed solution; In mixed solution, add equal-volume extract I, carry out the centrifugal 10min of 12000r/min, get supernatant liquor; In supernatant liquor, add equal-volume extract I, carry out the centrifugal 10min of 12000r/min, get supernatant liquor; In supernatant liquor, add equal-volume extract II, carry out the centrifugal 10min of 12000r/min, get supernatant liquor; In supernatant liquor, add the NaAC of 1/10 volume and the dehydrated alcohol of 2 times of volumes, mix gently, in-20 ℃ of precipitation 1h, then carry out the centrifugal 10min of 12000r/min; Solution after centrifugal is cleaned once with the ethanol that volume fraction is 70%, the more centrifugal 5min of 10000r/min, remove supernatant liquor, be precipitated; To precipitate natural air drying, and add 100 μ l sterilized waters and dissolve, obtain DNA solution, standby;
Step 1, DNA extraction: get sample 0.08g to be detected No. 27, after fully pulverizing, add 500 μ l lysates, be placed in 50 ℃ of water-baths and hatch 1h, obtain mixed solution; In mixed solution, add equal-volume extract I, carry out the centrifugal 10min of 12000r/min, get supernatant liquor; In supernatant liquor, add equal-volume extract I, carry out the centrifugal 10min of 12000r/min, get supernatant liquor; In supernatant liquor, add equal-volume extract II, carry out the centrifugal 10min of 12000r/min, get supernatant liquor; In supernatant liquor, add the NaAC of 1/10 volume and the dehydrated alcohol of 2 times of volumes, mix gently, in-20 ℃ of precipitation 1h, then carry out the centrifugal 10min of 12000r/min; Solution after centrifugal is cleaned once with the ethanol that volume fraction is 70%, the more centrifugal 5min of 10000r/min, remove supernatant liquor, be precipitated; To precipitate natural air drying, and add 100 μ l sterilized waters and dissolve, obtain DNA solution, standby;
Step 1, DNA extraction: get sample 0.12g to be detected No. 30, after fully pulverizing, add 500 μ l lysates, be placed in 60 ℃ of water-baths and hatch 2h, obtain mixed solution; In mixed solution, add equal-volume extract I, carry out the centrifugal 10min of 12000r/min, get supernatant liquor; In supernatant liquor, add equal-volume extract I, carry out the centrifugal 10min of 12000r/min, get supernatant liquor; In supernatant liquor, add equal-volume extract II, carry out the centrifugal 10min of 12000r/min, get supernatant liquor; In supernatant liquor, add the NaAC of 1/10 volume and the dehydrated alcohol of 2 times of volumes, mix gently, in-20 ℃ of precipitation 1h, then carry out the centrifugal 10min of 12000r/min; Solution after centrifugal is cleaned once with the ethanol that volume fraction is 70%, the more centrifugal 5min of 10000r/min, remove supernatant liquor, be precipitated; To precipitate natural air drying, and add 100 μ l sterilized waters and dissolve, obtain DNA solution, standby;
Embodiment 4
Step 1, DNA extraction: get sample 0.09g to be detected No. 34, after fully pulverizing, add 500 μ l lysates, be placed in 55 ℃ of water-baths and hatch 1.3h, obtain mixed solution; In mixed solution, add equal-volume extract I, carry out the centrifugal 10min of 12000r/min, get supernatant liquor; In supernatant liquor, add equal-volume extract I, carry out the centrifugal 10min of 12000r/min, get supernatant liquor; In supernatant liquor, add equal-volume extract II, carry out the centrifugal 10min of 12000r/min, get supernatant liquor; In supernatant liquor, add the NaAC of 1/10 volume and the dehydrated alcohol of 2 times of volumes, mix gently, in-20 ℃ of precipitation 1h, then carry out the centrifugal 10min of 12000r/min; Solution after centrifugal is cleaned once with the ethanol that volume fraction is 70%, the more centrifugal 5min of 10000r/min, remove supernatant liquor, be precipitated; To precipitate natural air drying, and add 100 μ l sterilized waters and dissolve, obtain DNA solution, standby;
Embodiment 5
Step 1, DNA extraction: get sample 0.11g to be detected No. 42, after fully pulverizing, add 500 μ l lysates, be placed in 58 ℃ of water-baths and hatch 1.8h, obtain mixed solution; In mixed solution, add equal-volume extract I, carry out the centrifugal 10min of 12000r/min, get supernatant liquor; In supernatant liquor, add equal-volume extract I, carry out the centrifugal 10min of 12000r/min, get supernatant liquor; In supernatant liquor, add equal-volume extract II, carry out the centrifugal 10min of 12000r/min, get supernatant liquor; In supernatant liquor, add the NaAC of 1/10 volume and the dehydrated alcohol of 2 times of volumes, mix gently, in-20 ℃ of precipitation 1h, then carry out the centrifugal 10min of 12000r/min; Solution after centrifugal is cleaned once with the ethanol that volume fraction is 70%, the more centrifugal 5min of 10000r/min, remove supernatant liquor, be precipitated; To precipitate natural air drying, and add 100 μ l sterilized waters and dissolve, obtain DNA solution, standby;
Negative comparative example
Negative control is carried out to agarose gel electrophoresis, and acquired results is shown in Fig. 1.
Positive comparative example
Positive control is carried out to agarose gel electrophoresis, and acquired results is shown in Fig. 1.
Interpretation of result: as shown in Figure 1, the negative contrast of c1, do not have two sections of digested one-tenth, and its position 1 is 1162bp; The positive contrast of c2, two sections of digested one-tenth, 10,27,30,34,42 are the sample number into spectrum chosen at random, have also digestedly become two sections, and long one section position 2 is 961bp, and short one section position 3 is 201bp.Through the detection of channel catfish species identification kit, the five tail samples of choosing at random all belong to the channel catfish species.By experimental result, can be found out, channel catfish species identification kit, very high to the specificity of the evaluation of channel catfish species, recall rate can reach 100%, is applicable to import and export the evaluation of channel catfish fully.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (5)
1. a channel catfish species identification kit, is characterized in that, comprises the reagent of following separation packing: lysate, extraction liquid I, extraction liquid II, PCR reaction solution, endonuclease reaction liquid, negative control and positive control;
Described lysate pH value is 8, comprises following component: 200mmol/L NaCl, 20mmol/LTris-HCl, 50mmol/L EDTA, 1%SDS, and all the other are water;
Described extraction liquid I comprises phenol, chloroform and primary isoamyl alcohol, and wherein the volume ratio of phenol, chloroform and primary isoamyl alcohol is 25:24:1;
Described extraction liquid II comprises chloroform and primary isoamyl alcohol, and wherein the volume ratio of chloroform and primary isoamyl alcohol is 24:1;
Described PCR reaction solution comprises following component: 10 * PCR Buffer, MgCl
2, dNTPs, upstream primer, downstream primer, Taq archaeal dna polymerase, all the other are ultrapure water; Concrete primer sequence is:
Upstream primer: 5 '-GTCGCCTCCGTACTGTATTTCTCC-3 ',
Downstream primer: 5 '-GGGTCCATCTTAACATCTTCAGTG-3 ';
Described endonuclease reaction liquid comprises 10 * Buffer and EcoR I;
Described positive control be after pcr amplification enzyme cut the channel catfish D-loop standard model of identifying;
Described negative control is the channel catfish D-loop standard model cut of enzyme not after pcr amplification.
2. channel catfish species identification kit according to claim 1, is characterized in that, in described PCR reaction solution, contains 10 * PCR Buffer2.5 μ l, MgCl
22.0 μ l, dNTPs2.0 μ l, upstream primer 0.5 μ l, downstream primer 0.5 μ l, Taq archaeal dna polymerase 0.5 μ l, and ultrapure water 16 μ l.
3. channel catfish species identification kit according to claim 1, is characterized in that, in described endonuclease reaction liquid, contains 10 * Buffer2.0 μ l and EcoR I 1.0 μ l.
4. according to the described channel catfish species of claims 1 to 3 any one identification kit, it is characterized in that, described lysate room temperature is deposited, and described extraction liquid I and extraction liquid II are stored in 4 ℃, and PCR reaction solution, endonuclease reaction liquid, negative control and positive control are stored in-20 ℃.
5. the using method of channel catfish species identification kit as claimed in claim 1, is characterized in that, comprises the following steps:
Step 1, DNA extraction:
1) get sample 0.08～0.12g to be detected, after fully pulverizing, add 500 μ l lysates, be placed in 50～60 ℃ of water-baths and hatch 1h～2h, obtain mixed solution;
2) in mixed solution, add equal-volume extract I, carry out the centrifugal 10min of 12000r/min, get supernatant liquor;
3) in supernatant liquor, add equal-volume extract I, carry out the centrifugal 10min of 12000r/min, get supernatant liquor;
4) in supernatant liquor, add equal-volume extract II, carry out the centrifugal 10min of 12000r/min, get supernatant liquor;
5) in supernatant liquor, add the NaAC of 1/10 volume and the dehydrated alcohol of 2 times of volumes, mix gently, in-20 ℃ of precipitation 1h, then carry out the centrifugal 10min of 12000r/min;
6) solution after centrifugal is cleaned once with the ethanol that volume fraction is 70%, the more centrifugal 5min of 10000r/min, remove supernatant liquor, be precipitated;
7) will precipitate natural air drying, and add 100 μ l sterilized waters and dissolve, obtain DNA solution, standby;
Step 2, pcr amplification:
1) the PCR reaction solution that thaws on ice, make it to dissolve fully, and the PCR reaction solution is joined in the PCR reaction tubes;
2) get the DNA solution 1 μ l that step 1 obtains, add in the PCR reaction tubes, mix;
3) the PCR reaction tubes is put into to the PCR instrument, response procedures is as follows: 95 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, annealing 30s, 72 ℃ are extended 1min, 35 circulations, last 72 ℃ are extended 10min, then are cooled to 12 ℃;
4) the PCR product of gained is placed on ice and can uses, or be placed in-20 ℃ standby;
Step 3, enzyme is cut evaluation:
(1) get step 2 and obtain PCR product 5 μ l, mix with endonuclease reaction liquid, 37 ℃ of incubation 2～3h, obtain enzyme and cut product;
(2) above-mentioned enzyme is cut to product and positive control and negative control and carried out agarose gel electrophoresis, take and identify whether testing sample is channel catfish according to electrophoresis result.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105603110A (en) * | 2016-03-28 | 2016-05-25 | 上海逍鹏生物科技有限公司 | Species identifying kit and species identifying method |
| CN108753988A (en) * | 2018-06-06 | 2018-11-06 | 江苏省淡水水产研究所 | A method of detection channel catfish HSP4 gene recessiveness causes albino mutation |
| CN109234407A (en) * | 2018-10-30 | 2019-01-18 | 中国计量大学 | A method of identification Du Shi rib Anchovy and red nose rib Anchovy |
| CN110628952A (en) * | 2019-10-25 | 2019-12-31 | 河海大学 | Primer, kit and method for rapidly detecting channel catfish herpesvirus and application of primer, kit and method |
-
2013
- 2013-07-24 CN CN201310313697.3A patent/CN103409515B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
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| BENJAPORN SOMRIDHIVEJ: "Characterization, polymorphism assessment, and database construction for microsatellites from BAC end sequences of channel catfish (Ictalurus punctatus)： A resource for integration of linkage and physical maps", 《AQUACULTURE》 * |
| MICAH SIMMONS: "Comparison of domestic and wild channel catfish (Ictalurus", 《AQUACULTURE》 * |
| 张卓娜: "斑点叉尾鮰NK-lysin 基因的cDNA 克隆及融合表达质粒构建", 《生物技术同胞》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105603110A (en) * | 2016-03-28 | 2016-05-25 | 上海逍鹏生物科技有限公司 | Species identifying kit and species identifying method |
| CN108753988A (en) * | 2018-06-06 | 2018-11-06 | 江苏省淡水水产研究所 | A method of detection channel catfish HSP4 gene recessiveness causes albino mutation |
| CN108753988B (en) * | 2018-06-06 | 2021-06-04 | 江苏省淡水水产研究所 | Method for detecting recessive whitening mutation of channel catfish HSP4 gene |
| CN109234407A (en) * | 2018-10-30 | 2019-01-18 | 中国计量大学 | A method of identification Du Shi rib Anchovy and red nose rib Anchovy |
| CN110628952A (en) * | 2019-10-25 | 2019-12-31 | 河海大学 | Primer, kit and method for rapidly detecting channel catfish herpesvirus and application of primer, kit and method |
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| Publication number | Publication date |
|---|---|
| CN103409515B (en) | 2014-08-20 |
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